The intake of nicotine results in the elevation of blood catecholamine levels, which is associated with increases in heart rate and blood pressure in humans and experimental animals. This increase in blood catecholamines is due partially to the nicotine-mediated increase in catecholamine secretion from the adrenal medulla. In order to prevent the depletion of the cellular stores of catecholamines during these periods of enhanced secretion, catecholamine biosynthesis is enhanced via the activation and induction of tyrosine hydroxylase (TH). The studies outlined in this proposal are designed to investigate the pharmacological and intracellular molecular mechanisms by which nicotine regulates TH in the rat adrenal medulla. The specific aims of the proposal are as follow: (1) to characterize pharmacologically the nicotine-mediated activation of TH and induction of TH-mRNA that occur in the adrenal medulla of rats treated with nicotine; (2) to determine whether the nicotine-mediated induction of adrenal TH-mRNA is due to the stimulation of the transcription rate of the TH gene; (3) to identify rat adrenomedullary nuclear proteins that interact with the 5'flanking region of the TH gene and that may be involved in the activation of the gene by nicotine; and (4) to determine whether tolerance develops to the nicotine-mediated activation of adrenal TH in chronically- treated rats. TH-mRNA levels will be measured by hybridization to a cloned cDNA probe. The transcription rate of the TH gene will be measured using a specific and sensitive nuclear run-on assay developed in my laboratory. Nuclear proteins that bind to he promoter regions of the TH gene will be measured by a gel shift assay using end-labeled restriction fragments isolated from a genomic clone that codes for the TH gene. THese measurements will be made using either control rats treated with saline or rats treated acutely or chronically for different periods of time and by different routes of administration with nicotine. These studies will increase our understanding of the mechanisms responsible for the regulation of adrenal TH by nicotine and thus, will shed light on the mechanisms by which nicotine elevates blood catecholamines and elicits the consequent cardiovascular effects of the drug.